A system for efficient assembly of HCV structural proteins into HCV-like particles (HCV-LPs) in insect cells has been developed in our laboratory. These noninfectious HCV-like particles have similar morphologic, serologic and biophysical properties as the putative virions isolated from HCV infected humans. In contrast to recombinant subunit vaccines, the viral proteins of HCV-like particles may be presented in a native, virion-like conformation and may therefore be superior in eliciting a protective humoral and cellular immune response. The humoral and cellular immunogenicity of the HCV-LP had previously been demonstrated in the mouse and baboon models. In addition, we demonstrated the immunogenicity and induction of protective immunity by HCV-LP in chimpanzees. Our study suggests that HCV-LP immunization induces strong HCV-specific cellular immune responses and confers partial protection against HCV challenge in the chimpanzee model. The purity of HCV-LP after our current purification procedure is only about 10%, which needs to be significantly improved if this approach is going to be developed clinically as a vaccine candidate. Based on what we know about the structural features of HCV envelope proteins in the viral particles, we engineered a histidineX6 tag at the N-terminus of E2 protein, which allows surface exposure of this tag. The histidine sequences should not interfere with the structural assembly of HCV and would facilitate affinity purification. Expression of this tagged E2 in the context of HCV-LP demonstrated feasibility of this approach and further work is being performed to improve HCV-LP purification. In an effort to improve and broaden the immunogenicity of HCV-LP, we report the generation of novel chimeric hepatitis C virus-like particles carrying HCV nonstructural protein sequences with T-cell epitopes important for induction of protective immunity. Six highly-conserved HCV CD8+ T-cell nonstructural epitopes associated with viral clearance in humans were synthesized as a polytope construct and fused to the C-terminus of the E2 protein of HCV-LP. Hydrophobicity of the signal peptide sequence in the C-terminus of E2 was reduced and the polytope sequence was confirmed. Chimeric HCV-LP carrying the HCV nonstructural polytope were amplified in insect cells, purified, and characterized biochemically, immunologically and by electron microscopy. The immunogenicity of the chimeric HCV-LP was tested in AAD transgenic mice expressing the human HLA-A2.1 molecule. Similar to HCV-LP, chimeric HCV-LP induced HCV-specific humoral and cellular immune responses against the core and envelope. In addition, chimeric HCV-LP elicited robust T-cell responses against the nonstructural epitopes. The chimeric HCV-LP polytope strategy substantially improved and broadened the immunogenicity of HCV-LP and holds promise as a vaccine candidate against HCV infection in humans.In addition, we are combining The HCV-LP approach with other modalities of immunization, such as plasmid DNA, in a prime-boost regimen. Therapy for hepatitis C virus (HCV) infection has advanced rapidly with the recent approval of two direct-acting antivirals, telaprevir and boceprevir, in combination with peginterferon and ribavirin. New antivirals with novel targets are still needed to further improve the treatment of hepatitis C. Previously reported screening methods for HCV inhibitors either limit to a virus-specific function or screen chemical libraries at a single dose which usually leads to high false-positive or -negative rates. We developed a quantitative high-throughput screen (qHTS) assay platform with a cell-based HCV infection system. The highly sensitive assay can be miniaturized to 1536-well format for screening of large-scale chemical libraries. All candidates are screened over a 7-concentration dose range to give IC50 values and dose-response curves. Using this assay format, we screened the library of pharmacologically active compounds (LOPAC). Based on the profile of dose-dependent curves of HCV inhibition and cytotoxicity, 22 compounds with adequate curves and IC50 <10 M were selected for validation. Using two additional independent assays, 17 of them demonstrated specific inhibition of HCV infection. Nine potential candidates with efficacy >70% and TC50 <30 M from these validated hits were further characterized for their target stages in the HCV life cycle. In this screen, we identified both known and novel hits with diverse structural and functional features targeting various stages of HCV life cycle. This pilot screen demonstrates that this assay system is highly robust and effective in identifying novel HCV inhibitors and can be readily applied to large-scale screen of small molecule libraries. We have completed a large-scale screen of chemical libraries consisting of MLSMR library of 400,000 compounds and 3000 NCGC pharmaceutical collection (NPC) library in collaboration with NCATS and identified several promising classes of compounds for further study. From the screen of the NPC library, chlorcyclizine HCl (CCZ), an approved drug for allergy symptoms, was identified and further characterized as a potent inhibitor of HCV infection. CCZ inhibited HCV infection in human hepatoma cells and primary human hepatocytes. The mode of action of CCZ is mediated by inhibiting early-stage of HCV infection, specifically targeting viral entry into host cells. The in vitro antiviral effect of CCZ is synergistic with other anti-HCV drugs, including ribavirin, interferon-&#945;, telaprevir, boceprevir, sofosbuvir, daclatasvir, and cyclosporin A without significant cytotoxicity, suggesting its potential in combination therapy of hepatitis C. In the mouse model, CCZ showed preferential liver distribution. In chimeric mice engrafted with primary human hepatocytes, CCZ significantly inhibited infection of HCV genotypes 1b and 2a without evidence of emergence of drug resistance. With its established clinical safety profile, affordability and a simple chemical structure for optimization, CCZ represents a promising candidate for drug repurposing and further development as a potent and highly accessible agent for treatment of HCV infection.